Application of Halberstadt lactobacillus jndm in relieving atopic dermatitis

Abstract

This invention provides the application of *Lactobacillus schreiberensis* JNDM from Harbin in alleviating atopic dermatitis, relating to the field of microbial technology. The metabiotic obtained from the fermentation of *Lactobacillus schreiberensis* JNDM from Harbin can significantly alleviate atopic dermatitis symptoms, reduce epidermal thickness, and restore skin barrier protein expression; it can also inhibit Th2-type inflammatory factors and serum IgE levels, and regulate systemic immune responses; the metabiotic obtained from the fermentation of *Lactobacillus schreiberensis* JNDM from Harbin remodels the gut microbiota structure through the "skin-gut axis," and can enrich... Alistipes The microbial community significantly increases the concentration of short-chain fatty acid butyric acid; it is also highly stable and safe to use, and can be applied to the preparation of drugs, food or daily chemical products for the prevention and treatment of atopic dermatitis.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application of Harbin Schleifer Lactobacillus JNDM in relieving atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is the most common chronic inflammatory skin disease, a multifactorial chronic or relapsing inflammatory skin condition. Symptoms include itchy, red, dry, and cracked skin. Some children with AD may even develop complications such as rhinophyma or asthma. The incidence of atopic dermatitis is high, with a prevalence of approximately 15-25% in children and 3-7% in adults. The pathogenesis of AD is complex, generally considered to involve abnormal immune responses, skin barrier dysfunction, and gut and skin microbiota dysbiosis. AD is highly correlated with the balance of T helper 1 (Th1), T helper 17 (Th17), and T helper 2 (Th2) immune regulation; Th2 responses are prevalent in the acute phase, while Th1 and Th17 responses dominate in the chronic phase.

[0003] For Alzheimer's disease (AD) patients treated with medication, different treatment methods are generally chosen depending on the skin lesions and the areas involved, such as topical corticosteroids, anti-infective therapy, topical immunomodulators, oral antibiotics, and biologics. However, this hormonal drug treatment is not only ineffective for patients with severe AD, but long-term use can also cause adverse reactions or side effects. Therefore, it is necessary to develop innovative therapies with reduced side effects to treat AD in children and adults.

[0004] The existing Chinese invention patent with publication number CN110616167A discloses Bifidobacterium that can alleviate atopic dermatitis and its application. Therefore, developing and exploring more treatment methods that can alleviate atopic dermatitis has important research significance and prospects. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of Harbin Schleifer Lactobacillus JNDM in alleviating atopic dermatitis, so as to enrich the treatment methods of atopic dermatitis and reduce the dependence and side effects of hormone drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of Harbin Schleifer Lactobacillus JNDM in the preparation of drugs for the prevention and treatment of atopic dermatitis.

[0007] This invention, through experiments, discovered a metabiotic derived from *Lactobacillus schreiformis* JNDM from Harbin. The active components are the fermentation broth and contents of this bacterium. Applied topically, it not only repairs the skin barrier but may also indirectly regulate the gut microbiota through the skin-gut axis, significantly alleviating symptoms of atopic dermatitis. *Lactobacillus schreiformis* JNDM from Harbin was deposited on December 11, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 64143). This strain has been published in Chinese Invention Patent Publication No. CN118421501A.

[0008] This invention provides a drug for the prevention and treatment of atopic dermatitis, comprising *Lactobacillus schleifera* JNDM or a metabiotic composed of fermentation product or cell contents obtained from the fermentation of *Lactobacillus schleifera* JNDM.

[0009] Preferably, the atopic dermatitis prevention and treatment drug is a fermentation broth or bacterial cell obtained by fermenting MRS liquid culture medium with Lactobacillus schleifera JNDM from Harbin at a temperature of 25~40℃.

[0010] Preferably, the fermentation is anaerobic fermentation.

[0011] Preferably, the fermentation broth is further centrifuged at a temperature of 2-6°C and a centrifugation speed of 10,000-15,000 rpm for 3-6 minutes.

[0012] Preferably, the bacterial cells are further subjected to pressure at a temperature of 2-6°C, the pressure being 600-1000 bar, and centrifuged to obtain lysed probiotics, with a centrifugation speed of 10,000-15,000 rpm for 3-6 minutes.

[0013] Furthermore, the aforementioned atopic dermatitis prevention and treatment drugs also include medically acceptable excipients.

[0014] This invention provides a gel formulation for the prevention and treatment of atopic dermatitis, comprising the following components: Harbin Schleifer Lactobacillus JNDM postbiotic 4.0~6.0 Carbomer 9400.5~1.0 Glycerin 4.0~8.0 Propylene glycol 3.0~7.0 Triethanolamine 0.2~1.0 EDTA-2Na 0.01~0.1 Phenoxyethanol 0.2~1.0 Remaining water.

[0015] This invention provides the application of Harbin Schleifer Lactobacillus JNDM in food, wherein the food is a health product or a dietary additive for food production.

[0016] This invention provides a health product containing *Lactobacillus schleifera* JNDM or a postbiotic composed of fermentation products or cell contents obtained from the fermentation of *Lactobacillus schleifera* JNDM.

[0017] This invention provides a dietary additive containing *Lactobacillus schleifera* JNDM or a postbiotic composed of fermentation products or cell contents obtained from the fermentation of *Lactobacillus schleifera* JNDM.

[0018] This invention provides the application of Harbin Schleifer Lactobacillus JNDM in skin care daily chemical products.

[0019] The present invention further provides a skin care daily chemical product containing *Lactobacillus schleifera* JNDM or a postbiotic composed of fermentation products or cell contents obtained from the fermentation of *Lactobacillus schleifera* JNDM.

[0020] Furthermore, the present invention specifically provides a facial mask essence, which is composed of the following components: Harbin Schleifer Lactobacillus JNDM postbiotic 4.0~6.0 Carbomer 9400.5~1.0 Glycerin 4.0~8.0 Butylene glycol 3.0~7.0 Sodium hyaluronate 0.01~0.5 Nicotinamide 0.1~3.0 Panthenol 0.2~1.0 EDTA-2Na 0.2~1.0 Phenoxyethanol 0.2~1.0 Remaining water.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The metabiotic obtained by fermentation of *Lactobacillus schreiberensis* JNDM in Harbin can significantly alleviate the symptoms of atopic dermatitis, reduce epidermal thickness, and restore skin barrier protein expression; it inhibits Th2-type inflammatory factors and serum IgE levels, and regulates systemic immune responses; the metabiotic obtained by fermentation of *Lactobacillus schreiberensis* JNDM in Harbin can reshape the gut microbiota structure through the "skin-gut axis" and can enrich... Alistipes The microbial community significantly increases the concentration of short-chain fatty acid butyric acid; it provides a safe and stable novel postbiotic preparation that can be used to prepare drugs, foods, or daily chemical products for the prevention and treatment of atopic dermatitis. Attached Figure Description

[0022] Figure 1 Comparison of cell scratch healing at 0 h and 24 h.

[0023] Figure 2 Comparison of cell scratch healing rates; Figure 2 The # indicates a statistically significant difference compared to the control group, and the * indicates a statistically significant difference compared to the model group (p<0.05 or p<0.01).

[0024] Figure 3 Gene FLG , LOR and IVL mRNA expression levels; Figure 3 The # indicates a statistically significant difference compared to the normal group, and the * indicates a statistically significant difference compared to the model group (p<0.05 or p<0.01).

[0025] Figure 4 Photographs of the backs of mice in each group after drug administration; Figure 4 A: Normal group; B: Model group; C: Positive drug group; D: Low-dose JNDM group; E: Medium-dose JNDM group; F: High-dose JNDM group; G: Commercially purchased strain group.

[0026] Figure 5 Results of atopic dermatitis scoring in mice.

[0027] Figure 6 Image of HE staining on mouse back skin; Figure 6 A: Normal group; B: Model group; C: Positive drug group; D: Low-dose JNDM group; E: Medium-dose JNDM group; F: High-dose JNDM group; G: Commercially purchased strain group.

[0028] Figure 7 Image of toluidine blue staining on the back skin of a mouse; Figure 7 A: Normal group; B: Model group; C: Positive drug group; D: Low-dose JNDM group; E: Medium-dose JNDM group; F: High-dose JNDM group; G: Commercially purchased strain group.

[0029] Figure 8 IL-4 , IL-5 , IL-13 , IL-33 , TSLP , GATA3 mRNA expression levels.

[0030] Figure 9 Distribution (a) and abundance (b) of gut microbiota.

[0031] Figure 10 Harbin Schleifer Lactobacillus JNDM postbiotic cytotoxicity test. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the experimental reagents and strains involved in the present invention are obtained commercially.

[0033] Example 1: Determination of the antioxidant capacity of *Lactobacillus schleifera* metabiotic from Harbin 1. Experimental Methods (1) Sample preparation To compare the antioxidant capacity of the present invention strain *Lactobacillus schreifer JNDM* with commercially available strains of the same species, *Lactobacillus plantarum*, and *Lactobacillus casei*, the specific experimental method is as follows: The present invention strain *Lactobacillus schreifer JNDM* and commercially available strains *Lactobacillus schreifer JNDM* (purchased from the China Industrial Microbial Culture Collection Center, product model or strain information CICC 6136), *Lactobacillus plantarum* (purchased from the China Industrial Microbial Culture Collection Center, product model or strain information CICC 10345), and *Lactobacillus casei* (purchased from the China Industrial Microbial Culture Collection Center, product model or strain information CICC 25035) were inoculated into MRS liquid medium at an inoculum of 3%, and cultured anaerobically at 37°C for 24 h to obtain fermentation cultures. The fermentation broth was centrifuged at 12,000 rpm for 5 min at 4℃, and the cell pellet was collected. After washing twice with sterile PBS, the PBS was diluted and resuspended to an OD600 nm value of 1.0. The cells were then homogenized using a high-pressure homogenizer at 4℃ (pressure 800 bar, 3 cycles). The homogenate was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected to obtain the lytic cells and basal ...

[0034] (2) Determination of DPPH free radical scavenging ability Take 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution, add 1 mL of the test sample, mix well, and react at room temperature in the dark for 30 min. Measure the absorbance A1 at a wavelength of 517 nm. Simultaneously, measure the absorbance A2 of 1 mL of sample mixed with 2 mL of anhydrous ethanol, and the absorbance A0 of 1 mL of distilled water mixed with 2 mL of DPPH solution. The DPPH scavenging rate is calculated using formula (I), with vitamin C (0.1 mg / mL) as a positive control.

[0035] DPPH clearance rate (%) = [1 - (A1 - A2) / A0] × 100%. (I) (3) Determination of hydroxyl radical scavenging ability To the reaction system, 1 mL of 9 mmol / L FeSO4, 1 mL of 9 mmol / L salicylic acid-ethanol solution, and 1 mL of the sample to be tested were added sequentially. Finally, 1 mL of 8.8 mmol / L H2O2 was added to start the reaction. The reaction was carried out in a water bath at 37℃ for 30 min, and the absorbance A1 was measured at 510 nm. The absorbance A0 was measured using distilled water instead of the sample. The hydroxyl radical scavenging rate was calculated as shown in equation (II).

[0036] Hydroxyl group scavenging rate (%) = (A0-A1) / A0 × 100% (II) 2. Experimental Results The experimental results are shown in Table 1. The results indicate that, compared with other similar or identical strains, the post-biotic lysate of *Lactobacillus schreiformis* JNDM from Harbin exhibited the strongest free radical scavenging ability. Specifically, in terms of DPPH free radical scavenging ability, JNDM achieved a scavenging rate of 72.5%, significantly higher than commercially available *Lactobacillus schreiformis* (45.3%), *Lactobacillus plantarum* (38.6%), and *Lactobacillus casei* (41.2%), approximately 1.6–1.9 times that of other strains. In terms of hydroxyl free radical scavenging ability, JNDM achieved a scavenging rate of 61.2%, also significantly higher than the other three control strains, demonstrating a clear advantage.

[0037] Table 1 Results of antioxidant capacity assay of strains

[0038] The above results indicate that the *Lactobacillus schreiformis* JNDM strain screened in this embodiment possesses excellent antioxidant activity, significantly superior to commercially available strains of the same species and other common probiotics. This strong antioxidant capacity helps to eliminate excess free radicals at the site of atopic dermatitis lesions, reducing oxidative stress damage, thereby providing a more favorable microenvironment for skin barrier repair and inflammation suppression. This is one of the important reasons why *Lactobacillus schreiformis* JNDM is used for skin barrier repair and inflammation suppression.

[0039] Example 2: Verification of Cell Barrier Repair Function 1. Experimental Methods (1) Cell culture and injury model establishment Commercially available HaCaT cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and then routinely cultured in a 37°C, 5% CO2 incubator. Cells were passaged after digestion when they reached 80% confluence.

[0040] HaCaT cells in the logarithmic growth phase were seeded into 6-well plates (2 × 10⁻⁶ cells / well). 5After culturing for 24 h, the cells were replaced with serum-free DMEM medium containing 10 ng / mL of TNF-α and IFN-γ for another 24 h to establish an atopic dermatitis-like cell injury model.

[0041] (2) Cell scratch repair experiment HaCaT cells were seeded into 12-well plates. After the cells grew into a confluent monolayer, a straight line was drawn in the center of each well using a sterile 200 μL pipette tip. The cells were then washed with PBS to remove any suspended cells. The following different treatment groups were then performed: Normal control group: supplemented with fresh DMEM medium without drugs; Model group: supplemented with DMEM medium containing 10 ng / mL TNF-α and IFN-γ, respectively; Positive control group: supplemented with DMEM medium containing 10 ng / mL TNF-α and IFN-γ, respectively. DMEM medium containing ng / mL dexamethasone (containing TNF-α+IFN-γ); JNDM post-biotic group (intervention group 1): DMEM medium containing 0.1% (v / v) Harbin Schleifer Lactobacillus JNDM cell lysate post-biotic (containing TNF-α+IFN-γ); Commercially available Harbin Schleifer Lactobacillus post-biotic group (intervention group 2): DMEM medium containing 0.1% (v / v) commercially available Harbin Schleifer Lactobacillus cell lysate post-biotic (containing TNF-α+IFN-γ); Lactobacillus plantarum post-biotic group (intervention group 3): DMEM medium containing 0.1% (v / v) Lactobacillus plantarum cell lysate post-biotic (containing TNF-α+IFN-γ); Lactobacillus casei post-biotic group (intervention group 4): DMEM medium containing 0.1% (v / v) Lactobacillus casei cell lysate post-biotic (containing TNF-α+IFN-γ).

[0042] Photographs were taken under an inverted microscope at 0 h and 24 h, and the width of each scratch was measured. The scratch healing rate was calculated using formula (III).

[0043] Healing rate (%) = (0 h width - 24 h width) / 0 h width × 100% (III) (3) Detection of skin barrier-related gene expression To further investigate the effects of different concentrations of post-biotic intervention, a new atopic dermatitis cell model was established without scratch treatment. The JNDM post-biotic dosage was set at 0.05%, 0.1%, 0.2%, and 0.4%, respectively. After 24 h of treatment with the same intervention method, cells were collected, and total RNA was extracted using the TRIzol method, reverse transcribed into cDNA, and barrier-related genes were detected by real-time quantitative PCR. FLG (Firmion) LOR (Hypothecium nitrate protein) IVL The mRNA expression level of (inner lining protein) is, in order to GAPDHThe primer sequences are shown in Table 2, and the sequence numbers are as shown in SEQ ID NO. 1~8. After amplification, the relative expression level of the target gene mRNA in the quantitative real-time PCR was calculated using the 2-ΔΔCt method, where ΔCt = target gene Ct value - internal reference gene Ct value, ΔΔCt = experimental group ΔCt - control group ΔCt, and 2-ΔΔCt represents the fold change in the expression of the target gene in the experimental group relative to the control group.

[0044] Table 2 Primer Sequences for Real-Time Quantitative PCR

[0045] 2. Experimental Results (1) Results of cell scratch test Cell scratch assay results are as follows Figure 1 and Figure 2 As shown, by Figures 1-2 It was found that, compared with the normal control group, the cell migration ability and scratch healing rate of the model group were significantly reduced, suggesting that inflammatory stimulation significantly inhibited the migration ability of keratinocytes. After different treatments, the cell migration ability of the positive control group was significantly improved. Among the JNDM post-modifier intervention groups, intervention group 1 showed the most significant promoting effect, with the highest scratch healing rate, significantly higher than the model group and exceeding the level of the normal control group. In contrast, the scratch healing rates of intervention groups 2, 3, and 4 were only slightly higher than the model group, and the difference was not significant compared with the model group, suggesting that their effect on improving cell migration ability was limited. The above results indicate that *Lactobacillus schleifera* JNDM post-modifier can significantly promote keratinocyte migration under specific conditions, with intervention group 1 showing the most significant effect.

[0046] (2) Gene FLG , LOR and IVL mRNA expression level results RT-qPCR results are as follows Figure 3 As shown, by Figure 3 The results showed that, compared with the normal group without modeling, the skin barrier-related genes in HaCaT cells of the model group stimulated with TNF-α / IFN-γ were significantly higher. FLG , LOR and IVL The mRNA expression levels were significantly reduced (P < 0.01), indicating that inflammatory stimulation successfully induced an atopic dermatitis-like cell injury model.

[0047] After 24 hours of postbiotic intervention with *Lactobacillus schleifera* JNDM from Harbin, the expression of the aforementioned barrier-related genes all showed varying degrees of recovery, exhibiting a dose-dependent increasing trend. When the postbiotic concentration reached 0.2% or higher, the expression of genes... FLG and LORThe expression level has basically recovered to or is close to the level of the normal group, while IVL Their expression has also improved significantly.

[0048] The above results indicate that the metabiotic derived from *Lactobacillus schleiferus* JNDM from Harbin in this invention can significantly upregulate skin barrier-related genes in keratinocytes. FLG , LOR and IVL The expression of [the substance] promotes the recovery of damaged skin barrier function and exhibits a significant dose-dependent repair effect.

[0049] Example 3: Therapeutic effect of DNFB-induced atopic dermatitis mouse model 1. Test Methods (1) Laboratory animals and grouping Fifty-six 6-week-old male BALB / c mice (SPF grade) were selected and randomly divided into groups of approximately eight mice each after one week of acclimatization. The groups included: a normal control group (NC group); a model group (M group); a positive control group (CRI group, criborone ointment); a low-dose JNDM postbiotic group (JNDM-L, 1%); a medium-dose JNDM postbiotic group (JNDM-M, 2.5%); a high-dose JNDM postbiotic group (JNDM-H, 5%); and a commercially available Harbin Schleifer lactobacillus postbiotic control group (Commercial group, 5%). All mice were housed in an SPF-grade animal facility at a temperature of 22±2℃ and a relative humidity of 50%±10%, with a 12-hour light-dark cycle and free access to food and water.

[0050] (2) Establishment and administration of atopic dermatitis model On days 2 and 5, mice were sensitized by applying 0.5% DNFB (acetone:olite = 4:1) to the skin on their backs (after hair removal). Starting from day 10, mice were challenged with 0.2% DNFB every other day for a total of 6 challenges. Four hours after each challenge, physiological saline was applied to the backs of the NC and M groups, criborone ointment was applied to the positive control group, and 50 μL / mouse was applied to the postbiotic treatment group. Administration continued until day 21. Specific administration and grouping methods are shown in Table 3.

[0051] Table 3 Grouping and Dosing Regimens of Experimental Animals

[0052] (3) Severity score of dermatitis Before each challenge and before sacrifice, the severity of dorsal dermatitis in mice was scored using a double-blind method, including four items: erythema / hemorrhagic rash, dryness, erosion, and epidermal shedding. Each item was scored from 0 to 4 points, and the total score was the sum of all items. Detailed scoring rules are shown in Table 4.

[0053] Table 4 Scoring criteria for the severity of dermatitis in mice

[0054] (4) Histological analysis Mice were sacrificed 24 hours after the last administration, and back skin was collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to H&E staining to observe epidermal thickness and inflammatory infiltration. Toluidine blue staining was used to observe mast cell infiltration.

[0055] (5) Detection of cytokines in skin tissue Total RNA was extracted from the back skin, and Th2 cytokines were detected by RT-qPCR. IL-4 , IL-5 , IL-13 , IL-33 , TSLP ) and transcription factors GATA3 The mRNA expression of the target is shown in Table 5, with sequence numbers SEQ ID NO.9~22.

[0056] Table 5. Primer sequences for mouse skin cytokines

[0057] (6) Collect whole blood from mice, separate serum, and use an ELISA kit (Elite) to detect the levels of total IgE and IFN-γ.

[0058] (7) 16S rRNA sequencing and short-chain fatty acid analysis of gut microbiota Cecal contents were collected, total DNA was extracted, and the 16S rRNA V3–V4 region was amplified and sequenced to analyze the microbial community composition and key differentially expressed microorganisms. Fecal samples were collected, and the contents of acetic acid, propionic acid, butyric acid, and valeric acid were determined by high-performance liquid chromatography.

[0059] 2. Experimental Results (1) JNDM post-biotic improves skin lesions in mice with atopic dermatitis Experimental results are as follows Figure 4 As shown, compared with the normal group, the mice in the DNFB treatment group exhibited obvious typical symptoms of atopic dermatitis on their backs, including erythema, dryness, desquamation, erosion, and crusting, indicating a significantly more severe degree of skin damage. After JNDM post-biotic intervention, the inflammatory symptoms on the backs of the mice were significantly alleviated, with the JNDM post-biotic treatment group showing the most significant improvement, showing a marked reduction in erythema and desquamation, and a significant decrease in the area of ​​skin lesions. The positive control drug, criborone ointment, also showed good therapeutic effects.

[0060] (2) JNDM post-biotic reduction of dermatitis severity score Dermatitis score results as follows Figure 5As shown in the results, the dermatitis scores of mice in the model group were significantly higher than those in the normal group, indicating that DNFB successfully induced an atopic dermatitis model. After JNDM post-biotic intervention, the dermatitis scores of all treatment groups decreased significantly, showing a dose-dependent decreasing trend. The 2.5% and 5% dose groups showed the most significant reduction (P < 0.01), with the 5% dose group showing better improvement than the positive control group, while the same commercially available strain did not show a significant improvement.

[0061] The above results indicate that JNDM postbiotics can effectively alleviate DNFB-induced atopic dermatitis symptoms in mice.

[0062] (3) Skin pathology sections H&E staining results are as follows Figure 6 As shown, the experimental results indicated that the normal group mice had intact skin structure, continuous epidermis without obvious defects, and no significant thickening of the papillary dermis, with only a small number of scattered inflammatory cell infiltrations. Compared with the normal group, the model group mice showed significant pathological changes in skin tissue, characterized by a large number of inflammatory cell aggregations and infiltrations, moderate to severe epidermal defects in some areas, and significant thickening of the papillary dermis, suggesting that DNFB successfully induced an atopic dermatitis-like inflammatory response. After intervention with the metagenerogenous agent, the pathological damage to mouse skin tissue was significantly improved. Compared with the model group, the treatment group showed a significant reduction in inflammatory cell infiltration, gradual recovery of epidermal structure, significant reduction in the degree of epidermal defects, and significant alleviation of papillary dermal thickening. Among them, the medium-dose and high-dose JNDM metagenerogenous agent groups showed more significant improvement, with their skin tissue structure approaching that of the normal group. However, the commercially available strains did not show significant improvement compared with the model group.

[0063] Toluidine blue staining results as follows Figure 7 As shown in the results, only a small number of scattered mast cells were observed in the skin tissue of normal mice. In the model group, the number of mast cells in the skin tissue was significantly increased, exhibiting obvious infiltration, with mast cell aggregation visible in some areas, indicating a significant inflammatory response in atopic dermatitis. After JNDM post-biotic intervention, mast cell infiltration in the mouse skin tissue was significantly reduced, and the number of mast cells was significantly lower than in the model group, mostly appearing in a scattered distribution. The improvement was more pronounced in the medium and high dose groups, while the commercially available bacterial strain showed no significant improvement compared to the model group.

[0064] The above results indicate that Harbin Schleifer Lactobacillus JNDM postbiotic can effectively inhibit atopic dermatitis-related inflammatory responses, reduce the infiltration of inflammatory cells and mast cells in skin tissue, and thus improve pathological damage to skin tissue.

[0065] (4) Effects of JNDM post-genetic agents on the expression of Th2 inflammatory factors in skin tissue RT-qPCR test results are as follows Figure 8As shown, the experimental results indicate that, compared with the normal group, the DNFB-induced model group mice had significantly higher levels of DNFB-induced abnormalities in their skin tissue. IL-4 , IL-5 , IL-13 , IL-33 , TSLP and transcription factors GATA3 The mRNA expression levels of the inflammatory factors were significantly increased, indicating a significant Th2-type immune response in the skin tissue of the model mice, demonstrating the successful establishment of the atopic dermatitis model. After intervention with *Lactobacillus schleifera* JNDM postbiotic, the expression levels of the aforementioned inflammatory factors in mouse skin tissue decreased to varying degrees, showing a dose-dependent trend. The 2.5% and 5% dose groups showed the most significant inhibitory effects. Simultaneously, the Th2 transcription factor GATA3 was significantly increased in the model group but significantly decreased in the JNDM postbiotic treatment group. Furthermore, compared with the commercially available *Lactobacillus schleifera* JNDM postbiotic control group, the JNDM postbiotic described in this invention showed a more significant effect in inhibiting the expression of related inflammatory factors and GATA3.

[0066] The above results indicate that *Lactobacillus schleifex* JNDM postbiotic from Harbin can alleviate atopic dermatitis-related inflammatory responses by inhibiting the expression of Th2-type inflammatory factors and regulating the related transcription factor GATA3, thus playing a significant role in improving skin inflammation.

[0067] (5) The effect of JNDM postbiotics on gut microbiota Alistipes Influence of genus abundance The effects of JNDM postbiotics on gut microbiota and abundance are as follows: Figure 9 As shown, by Figure 9 16S rRNA sequencing results showed that, at the genus level, there were significant differences in the gut microbiota composition among the experimental groups of mice. Alistipes The relative abundance of bacteria in different treatment groups varied significantly. Compared with the normal group, the gut microbiota of mice in the DNFB-induced atopic dermatitis model group was significantly higher. Alistipes The relative abundance of [a specific substance] was significantly reduced (P < 0.05), decreasing from 7.26% in the normal group to 2.54%, indicating a significant imbalance in the gut microbiota after the establishment of the atopic dermatitis model. Following intervention with *Lactobacillus schleifera* JNDM postbiotics in Harbin, Alistipes The relative abundance of *Lactobacillus schreiberensis* was significantly increased (P < 0.01). The relative abundances in the JNDM-1%, JNDM-2.5%, and JNDM-5% groups were 12.81%, 12.85%, and 16.37%, respectively, all significantly higher than the model group. Furthermore, the abundances showed an increasing trend with increasing dosage, with the JNDM-5% group showing the most significant increase. In contrast, the relative abundances in the commercially available Harbin Schleifer *Lactobacillus schreiberensis* postbiotic control group were significantly higher. AlistipesThe relative abundance was 5.20%, which was higher than that of the model group, but still significantly lower than that of the JNDM post-biotic administration group of this invention.

[0068] The above results indicate that the *Lactobacillus schleifera* JNDM postbiotic of this invention can significantly promote the growth of *Lactobacillus schleifera* in the intestine. Alistipes The proliferation of bacteria and the improvement of gut microbiota structure play an important role in regulating gut microbiota and alleviating the inflammatory response of atopic dermatitis.

[0069] (6) Results of fecal short-chain fatty acid content detection The contents of acetic acid, propionic acid, and butyric acid in the feces of mice in each group were detected by high performance liquid chromatography (HPLC), and the results are shown in Table 6. The results of fecal short-chain fatty acid detection showed that, compared with the normal group, the contents of acetic acid, propionic acid, and butyric acid in the feces of mice in the DNFB-induced atopic dermatitis model group were significantly reduced, decreasing from 48.6 μmol / g, 18.4 μmol / g, and 12.7 μmol / g to 29.3 μmol / g, 9.6 μmol / g, and 5.8 μmol / g, respectively. These differences were statistically significant, indicating that the intestinal microbial metabolic function was significantly affected after the establishment of the atopic dermatitis model.

[0070] After intervention with *Lactobacillus schleifera* JNDM postbiotics, the content of short-chain fatty acids in mouse feces significantly increased. The JNDM-1%, JNDM-2.5%, and JNDM-5% groups all increased the levels of acetic acid, propionic acid, and butyric acid to varying degrees, showing a dose-dependent trend. The JNDM-5% group showed the most significant increase, with acetic acid, propionic acid, and butyric acid levels reaching 52.8 μmol / g, 20.6 μmol / g, and 14.2 μmol / g, respectively. In contrast, although the commercially available *Lactobacillus schleifera* postbiotic control group showed some improvement in short-chain fatty acid content, the difference was not significant compared to the model group, and the overall increase was significantly lower than that of the JNDM postbiotic treatment group of this invention.

[0071] Table 6. Content of acetic acid, propionic acid and butyric acid in mouse feces

[0072] Note: * P<0.05, ** P<0.01, compared with the model group; # P<0.05, ## P<0.01, compared with the normal group.

[0073] The above results indicate that the *Lactobacillus schreiberensis* JNDM postbiotic from Harbin can promote the production of short-chain fatty acids in the gut and improve gut microbial metabolic function. This effect may be related to its ability to promote... Alistipes It is closely related to the proliferation of beneficial bacteria and the regulation of intestinal flora structure, thus playing an important role in alleviating the inflammatory response of atopic dermatitis.

[0074] Example 4: Cytotoxicity test of Lactobacillus schleifera JNDM post-genetic cytotoxicity in Harbin 1. Experimental Methods To evaluate the safety of *Lactobacillus schleiferus* JNDM post-biotic to skin cells, cytotoxicity was tested using human immortalized keratinocytes (HaCaT) as a model.

[0075] HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a constant temperature incubator at 37°C and 5% CO2. After the cells reached the logarithmic growth phase, they were digested with trypsin and seeded into 96-well plates at a density of approximately 5 × 10³ cells per well, and cultured for 24 h to allow the cells to adhere.

[0076] The original culture medium was then discarded, and culture medium containing different concentrations of *Lactobacillus schleifera* JNDM post-biotic was added for treatment. Six concentration gradients (1%, 2%, 4%, 6%, 8%, and 10%) were set up, with untreated cells serving as the control group. Each group had three replicates. After 24 h of treatment, cell viability was detected using a CCK-8 assay kit.

[0077] The specific procedure is as follows: Add 10 μL of CCK-8 working solution to each well, incubate for 1-2 h, and then measure the absorbance (OD value) at a wavelength of 450 nm. Cell viability is calculated using the following formula: Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%.

[0078] 2. Experimental Results Cytotoxicity test results as follows Figure 10 As shown, the experimental results indicate that within the concentration range of 1% to 10%, *Lactobacillus schleifera* JNDM post-biotic from Harbin did not exhibit significant toxicity to HaCaT cells. Compared with the control group, the cell viability in all treatment concentration groups remained above 90%, and cell viability was even slightly increased at some concentrations, but the overall difference was not significant.

[0079] The results showed that the Harbin Schleifer Lactobacillus JNDM post-genetic agent had good biocompatibility with skin keratinocytes in the concentration range of 1% to 10%, and did not significantly inhibit cell proliferation. This indicates that the post-genetic agent has good safety for skin application and can be used in the development of skin-related products or pharmaceutical formulations.

[0080] Example 5: Preparation of Harbin Schleifer Lactobacillus JNDM Postbiotic Gel Formulation 1. Composition of gel formulation The composition of the Harbin Schleifer Lactobacillus JNDM post-biotic gel preparation is shown in Table 7. The Harbin Schleifer Lactobacillus JNDM post-biotic is the post-biotic fermentation supernatant prepared in Example 1, which is obtained after centrifugation, filtration and concentration.

[0081] Table 7. Composition of Harbin Schleifer Lactobacillus JNDM Postbiotic Gel Formulation

[0082] 2. Preparation method of gel formulation The preparation steps of Harbin Schleifer Lactobacillus JNDM post-biotic gel formulation are as follows: (1) Carbomer matrix swelling Weigh out the amount of Carbomer 940 in the formula, slowly add it to an appropriate amount of purified water, and disperse it fully under magnetic stirring to make it swell evenly. Let it stand for hydration for 2 to 4 hours. In this example, it is preferred to obtain the Carbomer dispersion after 3 hours.

[0083] (2) Moisturizer mixture Glycerin and propylene glycol were added to a portion of purified water and mixed thoroughly under stirring. Then, EDTA-2Na and phenoxyethanol were added and stirring was continued until completely dissolved to obtain the excipient solution.

[0084] (3) Preparation of post-genetic agent solution Weigh out the prescribed amount of *Lactobacillus schleifera* JNDM postbiotic from Harbin, add an appropriate amount of purified water, and dissolve thoroughly under low-speed stirring to obtain a homogeneous postbiotic solution. To avoid degradation of active substances, the temperature throughout the process is controlled to not exceed 40℃; in this embodiment, 40℃ is preferred.

[0085] (4) Construction of gel system The excipient solution prepared in step (2) is slowly added to the carbomer dispersion obtained in step (1) and mixed evenly under continuous stirring. Then the post-generic solution prepared in step (3) is slowly added and stirring is continued to make the system uniform.

[0086] (5) pH adjustment and gel formation Triethanolamine was slowly added dropwise to the above mixture while continuously stirring. As the pH gradually increased, the carbomer neutralized and formed a transparent gel system. Finally, the pH of the system was adjusted to 6.0–6.5, preferably 6.3 in this example, to obtain a uniform and stable gel formulation.

[0087] (6) Degassing and filling The prepared gel is allowed to stand for 30-60 minutes to remove air bubbles, preferably 40 minutes in this embodiment. Then, it is filled into tubes or sealed containers under clean conditions and sealed for storage to obtain the Harbin Schleifer Lactobacillus JNDM post-biotic gel preparation.

[0088] Example 6: Stability test of Harbin Schleifer Lactobacillus JNDM post-biotic gel formulation 1. Experimental Methods The Harbin Schleifer Lactobacillus JNDM post-biotic gel preparation prepared in Example 5 was dispensed into sealed plastic tubes and placed under different storage conditions for stability testing.

[0089] Set the following storage conditions: Low temperature: 4℃; Room temperature: 25℃; Acceleration: 40℃.

[0090] Samples were taken at 0, 1, 2, 3 and 6 months of storage to observe the appearance of the gel formulation, and its pH value was measured using a pH meter and its viscosity change was measured using a rotational viscometer.

[0091] 2. Experimental Results (1) Visual observation During the stability test, the appearance of the gel formulation was observed, and its color, transparency, and whether layering or precipitation occurred were recorded.

[0092] The results showed that under various storage conditions, the JNDM post-biotic gel formulation maintained a uniform and transparent gel state without obvious stratification, precipitation, or color change, indicating that the formulation has good appearance stability.

[0093] (2) pH value change The pH changes of the gel formulation under different storage conditions are shown in Table 8. The results show that the pH value of the gel formulation changes little under different storage conditions, remaining consistently between 6.2 and 6.3, indicating that the gel system has good pH stability.

[0094]

[0095] Table 8. pH changes of JNDM post-biotic gel formulation under different storage conditions (3) Viscosity change The viscosity changes of the gel formulation under different storage conditions are shown in Table 9. The results indicate that the gel viscosity changed little during the 6-month storage period, and the system remained stable.

[0096] Table 9. Viscosity changes of JNDM post-biotic gel formulation under different storage conditions

[0097] (4) Experimental conclusions The stability test results show that the Harbin Schleifer Lactobacillus JNDM post-biotic gel formulation in this embodiment has good stability under different storage conditions. Its appearance, pH value and viscosity did not change significantly, indicating that the gel formulation has good storage stability.

[0098] Example 7: Preparation of Harbin Schleifer Lactobacillus JNDM Postbiotic Daily Chemical Products 1. Mask essence formula The composition of the Harbin Schleifer Lactobacillus JNDM post-biotic facial mask essence is shown in Table 10. The Harbin Schleifer Lactobacillus JNDM post-biotic is obtained from the fermentation supernatant prepared in Example 1 after centrifugation and filtration.

[0099] Table 10. Composition of the Essence Formula of Harbin Schleifer Lactobacillus JNDM Postbiotic Mask

[0100] 2. Preparation method of facial mask essence The preparation steps for Harbin Schleifer Lactobacillus JNDM Post-Shengyuan Facial Mask Essence are as follows: (1) Preparation of moisturizing system Referring to Table 10, add the prescribed amounts of glycerol and butylene glycol to an appropriate amount of purified water, mix thoroughly under stirring, then add EDTA-2Na, and continue stirring until completely dissolved.

[0101] (2) Addition of active ingredients Under continuous stirring, nicotinamide and panthenol were added sequentially, and stirring continued until completely dissolved. Sodium hyaluronate was then added, and the mixture was stirred at low speed to ensure thorough dispersion and swelling.

[0102] (3) Post-genetic addition Add Harbin Schleifer Lactobacillus JNDM postbiotic to the above solution and mix evenly under low-speed stirring, while controlling the system temperature to not exceed 40℃ to ensure the stability of the postbiotic active ingredients.

[0103] (4) Addition of anti-corrosion system Add phenoxyethanol to the system and continue stirring until completely homogeneous. Then add purified water to the formula volume and continue stirring for 20–30 minutes, preferably 25 minutes, to obtain a homogeneous and transparent mask essence.

[0104] 3. Mask preparation Non-woven fabric was selected as the mask carrier. The prepared mask essence was soaked into the mask fabric at a rate of 20-25 mL per sheet, allowing the mask fabric to fully absorb the essence. The mask fabric was then placed in an aluminum foil bag and sealed under clean, aseptic conditions to obtain the Harbin Schleifer Lactobacillus JNDM post-biotic mask preparation.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the solutions. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention based on the understanding of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of Harbin Schleifer Lactobacillus JNDM in the preparation of drugs for the prevention and treatment of atopic dermatitis.

2. A drug for the prevention and treatment of atopic dermatitis, characterized in that, This includes postbiotics composed of *Lactobacillus schleiferus* JNDM or fermentation products or cell contents obtained from the fermentation of *Lactobacillus schleiferus* JNDM.

3. The atopic dermatitis prevention and treatment drug according to claim 2, characterized in that, It also includes medically acceptable excipients.

4. A gel preparation for the prevention and treatment of atopic dermatitis, characterized in that, It consists of the following components: Harbin Schleifer Lactobacillus JNDM postbiotic 4.0~6.0; Carbomer 9400.5~1.0; Glycerin 4.0~8.0; Propylene glycol 3.0~7.0; Triethanolamine 0.2~1.0; EDTA-2Na 0.01~0.1; Phenoxyethanol 0.2~1.0; Remaining water.

5. The application of Harbin Schleifer Lactobacillus JNDM in food, characterized by, The food product is a health supplement or a dietary additive used in the production of the food.

6. A health product, characterized in that, It contains postbiotics composed of *Lactobacillus schleiferus* JNDM or fermentation products or cell contents obtained from the fermentation of *Lactobacillus schleiferus* JNDM.

7. A dietary additive, characterized in that, It contains postbiotics composed of *Lactobacillus schleiferus* JNDM or fermentation products or cell contents obtained from the fermentation of *Lactobacillus schleiferus* JNDM.

8. Application of Harbin Schleifer Lactobacillus JNDM in skin care daily chemical products.

9. A skincare daily chemical product, characterized in that, The skincare products contain *Lactobacillus schleifera* JNDM or postbiotics composed of ferments or cell contents obtained from the fermentation of *Lactobacillus schleifera* JNDM.

10. A facial mask essence, characterized in that, It consists of the following components: Harbin Schleifer Lactobacillus JNDM postbiotic 4.0~6.0; Carbomer 9400.5~1.0; Glycerin 4.0~8.0; Butanediol 3.0~7.0; Sodium hyaluronate 0.01~0.5; Nicotinamide 0.1~3.0; Panthenol 0.2~1.0; EDTA-2Na 0.2~1.0; Phenoxyethanol 0.2~1.0; Remaining water.

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